The current opioid prescription misuse epidemic has renewed interest in obtaining strong analgesic agents with decreased tolerance and addiction liability. Moreover, the high incidence of other side-effects with currently used opioid analgesics such as respiratory depression and constipation reduces their effectiveness in the pain clinic. Hence, there is a need either to develop new analgesics or to improve the clinical profile of existing medications. Morphine and related opioids exert their effects by acting at the orthosteric site on the mu-opioid receptor (MOR), i.e. the site where the endogenous opioid peptides bind. Recent advances in our knowledge of the structure of G-protein coupled receptors (GPCRs) has highlighted the possibility that GPCR function may be controlled by compounds binding at a separate, allosteric, site on the receptors. In this regard we have recently identified positive allosteric modulators (PAMs) that act at MOR (MOR-PAMs). These compounds increase the binding affinity of MOR agonists and the potency and/or maximal response to MOR agonists in vitro. Therefore, MOR-PAMs could possibly to be used as adjuncts to morphine and so reduce the level of opiate required to afford analgesia; thus producing the same functional analgesic response as a higher dose of morphine, but with reduced likelihood of the appearance of side-effects, including of tolerance and dependence. Perhaps more importantly, MOR-PAMs have the potential to be used alone to enhance the activity of endogenous opioid peptides. This would preserve the temporal and spatial characteristics of neuronal signaling and so avoid receptor down-regulation and other compensatory mechanisms that are induced by chronic MOR activation. In this application we seek to establish the value of allosteric modulation of MOR in vitro using an approach that involves investigation of the properties of allosteric modulation of MOR, structural biology to identify an allosteric binding site on MOR and development of improved allosteric probes. Detailed understanding of the actions of allosteric modulators of MOR will provide new information on mechanisms by which MOR function may be controlled and, together with the identification of high affinity probes, will pave the way for future drug development efforts of MOR modulators as analgesic adjunct drugs and/or novel analgesics.